Kynurenine 3-monooxygenase from Pseudomonas fluorescens: substrate-like inhibitors both stimulate flavin reduction and stabilize the flavin-peroxo intermediate yet result in the production of hydrogen peroxide.
Kynurenine 3-monooxygenase (KMO) is a flavin-dependent hydroxylase that catalyzes the conversion of l-kynurenine (l-Kyn) to 3-hydroxykynurenine (3OHKyn) in the pathway for tryptophan catabolism. KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. We have investigated the reductive and the oxidative half-reactions of a stable form of KMO from Pseudomonas fluorescens (KMO). The binding of l-Kyn by the enzyme is relatively slow and involves at least two reversible steps. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of approximately 2.5 x 10(3) when l-Kyn is bound. The rate of reduction of the KMO.l-Kyn complex is 160 s(-1), and the K(d) for the NADPH complex is 200 microM with charge-transfer absorption bands for the KMO(RED).l-Kyn.NADP(+) complex accumulating after reduction. The reduction potential of KMO is -188 mV and is unresponsive to the addition of l-Kyn or other inhibitory ligands. KMO inhibitors whose structures are reminiscent of l-Kyn such as m-nitrobenzoylalanine and benzoylalanine also stimulate reduction of flavin by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. A spectrum consistent with that of a peroxyflavin species accumulates and then decays to yield the oxidized enzyme. This species then undergoes minor spectral changes that, based on flavin difference spectra defined in the presence of 3OHKyn, can be correlated with product release. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine also shows the accumulation of a peroxyflavin species that then decays to yield hydrogen peroxide without hydroxylation.
Crozier-Reabe
Karen R
KR
Department of Chemistry and Biochemistry, University of Wisconsin, 3210 North Cramer Street, Milwaukee, Wisconsin 53211-3029, USA.
Phillips
Robert S
RS
Moran
Graham R
GR
D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D005415:Flavins / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007700:Kinetics; D007737:Kynurenine / Q000378:metabolism / Q000494:pharmacology; D050601:Kynurenine 3-Monooxygenase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008024:Ligands; D010084:Oxidation-Reduction / Q000187:drug effects; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D012965:Sodium Chloride / Q000494:pharmacology
D004791:Enzyme Inhibitors; D005415:Flavins; D008024:Ligands; D007737:Kynurenine; D012965:Sodium Chloride; D006861:Hydrogen Peroxide; D050601:Kynurenine 3-Monooxygenase